Inhibitors of positive strand rna viruses

ABSTRACT

Methods of treating a disease caused by a positive strand RNA virus. The methods include administering to a subject in need thereof an effective amount of a compound of Formula I.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 17/369,589, filed on Jul. 7, 2021, which claims the benefit ofpriority based on U.S. Provisional Application No. 63/049,140, filedJul. 8, 2020. The entire content and disclosure of both applications areincorporated herein by reference in their entirety.

BACKGROUND

Positive strand RNA viruses include pathogens such as Zika virus, Denguevirus, human coronavirus OC43 (“HCoV-OC43”), human coronavirus 229E(“HCoV-229E”), and severe acute respiratory syndrome coronavirus 2(“SARS-CoV-2”).

Zika virus caused the widespread epidemic of Zika fever in 2015 and2016. It can be transmitted from a pregnant woman to her fetus, causingmicrocephaly and other severe brain anomalies in infants. Currently,there is no specific medicine or vaccine for Zika virus.

Dengue virus spreads through the bite of an infected Aedes speciesmosquito, causing Dengue fever. It is common in more than 100 countriesand infects millions of people worldwide each year. Only one vaccine,i.e., Dengvaxia®, has been approved so far for people who have alreadyhad dengue fever at least once. Preventing mosquito bites andcontrolling mosquito population remain the main methods for fightingDengue virus.

HCoV-OC43 and HCoV-229E, viruses responsible for the common cold, areassociated with upper respiratory tract infections. They can also causesevere lower respiratory tract illnesses, including bronchiolitis,bronchitis, croup, and pneumonia, primarily in infants andimmunocompromised patients. Medicines are available only to reduce painand fever. Vaccines have not been commercialized to protect peopleagainst these two human coronaviruses.

SARS-CoV-2 caused the COVID-19 pandemic, leading to more than 3.9million of deaths worldwide. To contain the spread of the pandemic, manycountries implemented year-long non-pharmaceutical interventions such asstay-at-home orders, curfews, and quarantines, sending the globaleconomy into a recession. The US Food and Drug Administration hasapproved one medicine, remdesivir, to treat COVID-19. Studies show thatremdesivir is effective in only a small portion of patients, allowingthem to recover faster. Other potential treatments include repurposedmedicines, e.g., baricitinib, dexamethasone, ciclesonide, chloroquine,and hydroxychloroquine. None of the treatments has high efficacy.

There is an unmet need to develop an efficient treatment for infectionscaused SARS-CoV-2, Dengue virus, Zika virus, HCoV-OC43, and HCoV-229E.Particularly, a potent treatment for COVID-19 is urgently demanded.

SUMMARY

To address the above need, certain compounds have been identified thatsurprisingly inhibit positive strand RNA viruses including Dengue virus,Zika virus, HCoV-OC43, HCoV-229E, and SARS-CoV-2.

Accordingly, one aspect of this invention relates to a method oftreating a disease caused by a positive strand RNA virus. The methodincludes the steps of identifying a subject suffering from the diseaseand administering to the subject an effective amount of a compound ofFormula I below:

In this formula, (1) each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅, independently, is H, halo, alkyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, OH, alkoxy, carbonyloxy, oramino; (2) each of R₁₆ and R₁₇, independently, is H, halo, alkyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, OH, alkoxy, carbonyloxy, oramino, or R₁₆ and R₁₇ together are a single bond; (3) each of

and

, independently, is a single bond or a double bond; and (4) n is 1, 2 or3. When

between X and Y is a single bond, X is C═O or CR′R″ and Y is N or N⁺→O⁻,in which each of R′ and R″, independently, is H, halo, alkyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, OH, alkoxy, or amino. When

between X and Y is a double bond, X is CR′, Y is N+, and a counterioncoexists in the compound, R′ being defined above.

Preferably, each of R₁, R₅, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅ isH; each of R₂, R₃, R₆, and R₇, independently, is alkoxy (e.g., C₁-C₆alkoxy, methoxy, and ethoxy); R₄ is H or alkoxy (e.g., C₁-C₆ alkoxy,methoxy, and ethoxy); R₁₆ and R₁₇ together are a single bond; X is CH₂;and Y is N or N+→O—; each of

and

is a single bond; and n is 1 or 2.

The structures of three exemplary compounds of Formula I, i.e., Compound1, Compound 4, and Compound 6, shown below:

The positive strand RNA virus can be a flavivirus (e.g., Zika virus,Dengue virus) or a coronavirus (e.g., SARS-CoV-2, HCoV-OC43, andHCoV-229E).

Another aspect of this invention relates to a treatment method includingthe steps of identify a subject having a disease caused by a positivestrand RNA virus and administering to the subject an effective amount ofa compound of Formula II:

In this formula, (1) each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, andR₁₀, independently, is H, alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, halo, nitro, cyano, —OR^(a),OC(O)R^(a), —C(O)OR^(a), —NR^(a)R^(b), —NR^(a)C(O)R^(b), or—C(O)NR^(a)R^(b), each of R^(a) and R^(b), independently, being H,alkyl, aryl, heteroaryl, cycloalkyl, or hetercycloalkyl; (2) X

Y, together, is C(R′)(R″)—N or CR′═N⁺, in which R′ is H, alkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halo, nitro, orcyano, and R″ is H, alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, halo, nitro, cyano, —OR^(c), or—OC(O)R^(c), R^(c) being H, alkyl, aryl, heteroaryl, cycloalkyl, orhetercycloalkyl; (3) A is H, alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; and (4)

is a single bond or a double bond.

Preferably, each of R₁, R₄, R₅, and R₈ is H; R₂ is H or alkoxy (e.g.,C₁-C₆ alkoxy, methoxy, and ethoxy); each of R₃, R₆, and R₇,independently, is alkoxy (e.g., C₁-C₆ alkoxy, methoxy, and ethoxy); R₉is H or alkyl (e.g., C₁-C₆ alkyl, methyl, ethyl, propyl, and isopropyl),R₁₀ is H or OH; X is CH₂; Y is N; A is alkyl (e.g., C₁-C₆ alkyl, methyl,ethyl, propyl, and isopropyl); and

is a single bond.

Exemplary compounds of Formula II include Compounds 2, Compound 3,Compound 5, Compound 7, Compound 8, and Compound 9, the structures ofwhich follow:

The term “alkyl” herein refers to a straight or branched hydrocarbongroup, containing 1-20 (e.g., 1-10 and 1-6) carbon atoms. Examplesinclude methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, andt-butyl. Alkyl includes its halo substituted derivatives, i.e.,haloalkyl, which refers to alkyl substituted with one or more halogen(chloro, fluoro, bromo, or iodo) atoms. Examples includetrifluoromethyl, bromomethyl, and 4,4,4-trifluorobutyl. The term“alkoxy” refers to an —O-alkyl group. Examples include methoxy, ethoxy,propoxy, and isopropoxy. Alkoxy includes haloalkoxy, referring to alkoxysubstituted with one or more halogen atoms. Examples include —O—CH₂Cland —O—CHClCH₂Cl.

The term “cycloalkyl” refers to a saturated and partially unsaturatedmonocyclic, bicyclic, tricyclic, or tetracyclic hydrocarbon group having3 to 12 carbons. Examples of cycloalkyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heterocycloalkyl” refers to a nonaromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having one or more heteroatoms (e.g., O, N, P, and S). Examplesof heterocycloalkyl groups include, but are not limited to, piperazinyl,imidazolidinyl, azepanyl, pyrrolidinyl, dihydrothiadiazolyl, dioxanyl,morpholinyl, tetrahydropuranyl, and tetrahydrofuranyl.

The term “aryl” refers to a 6-carbon monocyclic, 10-carbon bicyclic,14-carbon tricyclic aromatic ring system wherein each ring may have 1 to5 substituents. Examples of aryl groups include phenyl, naphthyl, andanthracenyl. The term “arylene” refers to bivalent aryl. The term“aralkyl” refers to alkyl substituted with an aryl group.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system havingone or more heteroatoms (e.g., O, N, P, and S). Examples includetriazolyl, oxazolyl, thiadiazolyl, tetrazolyl, pyrazolyl, pyridyl,furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl,indolyl, thiazolyl, and benzothiazolyl. The term “heteroaryl alkyl”refers to an alkyl group substituted with a heteroaryl group.

The terms “halo” refers to a fluoro, chloro, bromo, or iodo radical. Theterm “amino” refers to a radical derived from amine, which isunsubstituted or mono-/di-substituted with alkyl, aryl, cycloalkyl,heterocycloalkyl, or heteroaryl. The term “alkylamino” refers toalkyl-NH—. The term “dialkylamino” refers to alkyl-N(alkyl)-.

Alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, alkoxy, and aryloxy mentioned herein include bothsubstituted and unsubstituted moieties. Examples of substituentsinclude, but are not limited to, halo, hydroxyl, amino, cyano, nitro,mercapto, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl,carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfonamido,alkyl, alkenyl, alkynyl, alkyloxy, aryl, heteroaryl, cycloalkyl, andheterocycloalkyl, in which alkyl, alkenyl, alkynyl, alkyloxy, aryl,heteroaryl cycloalkyl, and heterocycloalkyl may further substituted.

Compounds of Formula I or Formula II can include an anion. Examples ofan anion include Cl⁻, Br⁻, I⁻, SO₄ ²⁻, PO₄ ³⁻, ClO₄ ⁻, CH₃CO₂ ⁻, andCF₃CO₂ ⁻.

The term “compound”, when referring to a compound of Formula I orFormula IL, also covers its salts, solvates, and prodrugs. A salt can beformed between an anion and a positively charged group (e.g., amino) ona compound; examples of a suitable anion include chloride, bromide,iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate,trifluoroacetate, acetate, malate, tosylate, tartrate, fumurate,glutamate, glucuronate, lactate, glutarate, and maleate. A salt can alsobe formed between a cation and a negatively charged group; examples of asuitable cation include sodium ion, potassium ion, magnesium ion,calcium ion, and an ammonium cation such as tetramethylammonium ion.Further, a salt can contain quaternary nitrogen atoms. A solvate refersto a complex formed between an active compound and a pharmaceuticallyacceptable solvent. Examples of a pharmaceutically acceptable solventinclude water, ethanol, isopropanol, ethyl acetate, acetic acid, andethanolamine. A prodrug refers to a compound that, after administration,is metabolized into a pharmaceutically active drug. Examples of aprodrug include esters and other pharmaceutically acceptablederivatives, which, upon administering to a subject, are capable ofproviding active compounds of this invention.

The details of the invention are set forth in the drawings, thedefinitions, and the detailed description below. Other features,objects, and advantages of the invention will be apparent from thefollowing actual examples and claims.

BRIEF DESCRIPTION OF THE DRAWING

The description below refers to the accompanying drawing:

FIG. 1 shows antiviral activities against HCoV-OC43 by three tylophorinecompounds, i.e., Compounds 1-3, at concentrations in the range of 0 to300 nM.

FIG. 2 shows antiviral activities against HCoV-OC43 by Compounds 1-3 atconcentrations in the range of 0 to 50 nM.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed above, tylophorine (i.e., Compound 1) and its derivativesare used to treat diseases caused by a positive strand RNA virus.

Tylophorine, C₂₄H₂₇NO₄, is a major alkaloid contained in roots andleaves of tylophora indica, a climbing plant originally established inthe eastern and southern India. Tylophora indica is widely used in folkmedicines for allergies, asthma, cancers, coughing, joint disorders(rheumatoid arthritis), etc.

Tylophorine was first extracted from the plant as early as 1935. SeeRatnagiriswaran et al., Indian J. Med. Res. 22(3), 433-441 (1935).Biologically efficacious tylophorine was extracted from tylophora indicaby acid extraction and solvent distillation utilizing methanol, ethylacetate, and chloroform almost four decades later. See Rao et al., F. J.Pharma. Sci. 60(11), 1725-26 (1971). The structure of tylophorine waselucidated by different spectroscopic techniques. See Govindachari etal., Proc. Indian Acad. Sci. 3, 114 (2002). Tylophorine, having amolecular weight of 393.48, is a secondary metabolite containingorganonitrogen heterocyclic and organic heteropentacyclic compounds. ItsIUPAC name is(13as)-2,3,6,7-tetramethoxy-9,11,12,13,13a,14-hexahydrophenanthro[9,10-f]indolizine. See the Summary section above for its chemical structure.

Tylophorine possesses several properties, such as immunosuppressive,antitumor, antifeedant, antibacterial, antifungal, antiamoebic, diureticand hepatoprotective activities. In addition, it provides positivestimulation to adrenal cortex.

Biotechnological production of tylophorine is fulfilled by inducinghairy roots mediated by Agrobacterium rhizogenes (A4 strain). Chemicalsynthesis of tylophorine, on the other hand, can be achieved using afive-step process via a nitrile stabilized ammonium intermediate. SeeLahm et al., J. Org. Chem. 77, 6620-23 (2012). Tylophorine iscommercially available as yellow solid. Suppliers include Alfa Chemistry(Ronkonkoma, New York), Glixx Laboratories Inc. (Hopkinton,Massachusetts), and MedKoo Biosciences Inc. (Morrisville, NorthCarolina).

Tylophorine and its derivatives have been used for treating cancers andother disorders. See U.S. Pat. Nos. 7,652,027, 9,216,977 and 8,486,959,and US Patent Application Publication 2011/0201637. Derivatives aretypically prepared via chemical syntheses. See, e.g., U.S. Pat. No.8,486,959, US Patent Application Publication 2011/0201637, Yang et al.,Antivirai Res. 88, 160-168 (2010); and Lee et al., J. Med. Chem. 55,10363-77 (2012).

Compounds of Formula I or Formula II can be prepared by conventionalmethods, e.g., procedures provided in the references cited above. Theirantiviral activities are then evaluated using known methods such asthose described in actual examples below.

Some compounds of this invention contain a non-aromatic double bond orone or more asymmetric centers. Each of them occurs as a racemate or aracemic mixture, a single R enantiomer, a single S enantiomer, anindividual diastereomer, a diastereometric mixture, a cis-isomer, or atrans-isomer. Compounds of such isomeric forms are within the scope ofthis invention. They can be present as a mixture or can be isolatedusing chiral synthesis or chiral separation technologies.

A compound of Formula I or Formula II is preferably formulated into apharmaceutical composition containing a pharmaceutical carrier. Thecomposition is then given to a subject in need thereof to treat aCisd2-insufficient associated disorder or protect againstdoxorubicin-induced cardiotoxicity.

To practice the method of the present invention, a composition havingone or more of the above-described tylophorine compounds can beadministered parenterally, orally, nasally, rectally, topically, orbuccally.

The term “parenteral” as used herein encompasses subcutaneous,intracutaneous, intravenous, intraperitoneal, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection of a sterile injectablecomposition. Indeed, the term refers to any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution, and isotonic sodiumchloride solution. In addition, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordi-glycerides). Fatty acid, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil and castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long chain alcohol diluent or dispersant,carboxymethyl cellulose, or similar dispersing agents. Other commonlyused surfactants such as Tweens and Spans or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.Oral solid dosage forms can be prepared by spray dried techniques; hotmelt extrusion strategy, micronization, and nano milling technologies.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as a solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. A composition havingan active compound can also be administered in the form of suppositoriesfor rectal administration.

The carrier in the pharmaceutical composition must be “acceptable” inthe sense that it is compatible with the active ingredient of thecomposition (and preferably, capable of stabilizing the activeingredient) and not deleterious to the subject to be treated. One ormore solubilizing agents can be utilized as pharmaceutical excipientsfor delivery of an active compound. Examples of other carriers includecolloidal silicon oxide, magnesium stearate, cellulose, sodium laurylsulfate, and D&C Yellow #10.

The term “treating” refers to application or administration of thecompound to a subject with the purpose to cure, alleviate, relieve,alter, remedy, improve, or affect the disease, the symptom, or thepredisposition. “An effective amount” refers to the amount of thecompound which is required to confer the desired effect on the subject.Effective amounts vary, as recognized by those skilled in the art,depending on route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatments such as use ofother active agents. Dosage levels of a compound of Formula I or FormulaII are of the order of 0.01 mg/kg body weight to 500 mg/kg body weight(e.g., 0.05 mg/kg body weight to 300 mg/kg body weight, 0.1 mg/kg bodyweight to 200 mg/kg body weight, and 1 mg/kg body weight to 100 mg/kgbody weight) per day. The specific dose level for a particular patientwill depend upon a number of factors including age, body weight, generalhealth, sex, diet, time of administration, rate of excretion, and theseverity of the disorder. To enhance the therapeutic efficiency, thecompound can be administered concomitantly with one or more of otherorally active antiviral compounds.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific examples are, therefore, tobe construed as merely illustrative, and not limitative of the remainderof the disclosure in any way whatsoever.

All publications, including patent documents, cited herein areincorporated by reference in their entirety.

EXAMPLES

Tylophorine and eight tylophorine derivatives, i.e., Compounds 1-9, weretested and unexpectedly showed antiviral activities against positivesingle stranded RNA viruses, i.e., SARS-CoV-2, HCoV-OC43, HCoV-229E,Zika virus, and Dengue virus.

Example 1: Inhibition of SARS-CoV-2

An immunofluorescence assay (“IFA”) and a plaque assay were performed toevaluate inhibition of SARS-CoV-2.

SARS-CoV-2 Immunofluorescence Assay

Vero E6 cells derived from BCRC #60476 (Bioresource Collection andResearch Center, Hsinchu, Taiwan) were treated with each compound at anindicated concentration for 1 hour at 37° C. The cells were adsorbedwith SARS-CoV-2 viruses (TCDC #4, National Taiwan University, Taipei,Taiwan, ROC) at multiplicity of infection (“MOI”) of 0.01 for 1 hour at37° C. After virus adsorption, the cells were washed withphosphate-buffered saline (“PBS”). A fresh medium containing thecompound was added at an indicated concentration. The resultant mixturewas incubated for 2 days. The cells were fixed with 4% paraformaldehydeand permeabilized with a 0.5% Triton™ X-100 detergent solution (ThermoFisher Scientific, Waltham, MA). Subsequently, they were stained with ananti-SARS-CoV-2 N protein antibody and anti-human IgG-488 (in green).The nuclei of the cells were counter stained with4′,6-diamidino-2-phenylindole (“DAPI”, Thermo Fisher Scientific, MA,USA). The N protein expression was measured using a high-content imageanalysis system (Molecular Devices, San Jose, CA). The cell viabilitywas determined by Cell Counting Kit-8 (Sigma-Aldrich, St. Louis, MO)EC₅₀ and CC₅₀ values were calculated by Prism software. The term “EC₅₀”refers to the half maximal effective concentration of a compound atwhich a virus is inhibited by 50%. The term “CC₅₀” refers to the 50%cytotoxic concentration of a compound at which the cell viability isreduced by 50%. Both EC₅₀ and CC₅₀ are measured in molar units, e.g.,mol/L (“M”), μmol/L (“μM”), and nmol/L (“nM”).

The results are shown in Table 1 below. Compounds 1, 2, and 3 each had ahigh inhibitory activity against SARS-CoV-2, having a very low EC₅₀ inthe range of 9 nM to 77 nM.

SARS-CoV-2 Plaque Assay

The assay was performed in triplicate in 24-well tissue culture plates.Vero E6 cells were seeded in Dulbecco's modified Eagle's medium (“DMEM”)with 10% Fetal Bovine Serum (“FBS”, Biological Industries, Kibbutz,Israel) and antibiotics one day before infection. SARS-CoV-2 viruseswere added to the cell monolayer and allowed to sit for 1 hour at 37° C.After viruses were removed, the cell monolayer was washed once with PBSbefore covering with media containing agarose or methylcellulose for 5-7days. The cells were fixed with 3.7% formaldehyde overnight followed byremoval of overlay media. They were then stained with crystal violet tocount the plaque-forming units (“PFU”). The percentage of inhibition wascalculated as [1−(V_(D)/V_(C))]×100%, where V_(D) and V_(C) refer to thevirus titer in the presence and absence of a test compound,respectively.

Inhibition of SARS-CoV-2

Compounds 1-3 were examined for their inhibitory activity againstSARS-CoV-2 in Vero E6 cells. Cytopathic effects (“CPE”) of SARS-CoV-2infected Vero E6 cells, with treatment of compounds in 2-fold dilutionat a series of 12 concentrations, were visualized. EC₅₀ and CC₅₀ valueswere calculated. See Table 1 below.

TABLE 1 SARS-CoV-2 inhibition EC₅₀ (nM) Compound CPE IFA-1 IFA-2 Plaqueassay-1 Plaque assay-2 1 78 64 76.8 nd nd 2 2.5 9.1 13.9 10.8 13.8 3 2031.6 31.9 nd nd CPE: cytopathic effect by visualization.

Compounds 1-3 each had a high anti-SARS-CoV-2 activity as shown by theCPE results. In addition, the IFA assay showed results comparable tothose from the CPE results. Surprisingly, Compounds 1-3 each had a highpotency of inhibiting SARS-CoV-2. Among them, Compound 2 had the highestantivirus activity. The plaque assay was performed four times to measurethe potency of Compound 2 against SARS-CoV-2 in infected Vero E6 cells.In all four measurements, Compound 2 had an EC₅₀ value in the range of11-14 nM, which was in consistence with those obtained from thecytopathic effect assay and the IFA assay shown in Table 1 above.

Example 2: Inhibition of HCoV-OC43

Compounds 1-6 were tested for their inhibitory activities against humancoronavirus HCoV-OC43 (betacoronavirus) using an IFA assay, whichtargeted HCoV-OC43 nucleocapsid (“N”) protein.

Human colon adenocarcinoma cell line HCT-8 (ATCC@ CCL-244™, AmericanType Culture Collection, Manassas, VA, USA) was obtained from AmericanType Culture Collection (“ATCC”). It was established as stock at earlypassage to ensure cell line-specific characteristics. HCoV-OC43 viruses(ATCC@VR1558™, American Type Culture Collection, Manassas, VA, USA) weregrown and propagated in HCT-8 cells cultured with DMEM and 2% FBS(Biological Industries, Kibbutz, Israel). The cells were seeded in a96-well plate and then cultured in a DMEM medium containing 2% FBS.Subsequently, they were pretreated with one of Compounds 1-6 at one offive predetermined concentrations in a 5-fold dilution for 1 hour priorto HCoV-OC43 virus infection at an MOI of 0.05. The resultantsupernatant at the 72 d.p.i. were subjected to an end-point assay and aTCID50 determination after 6 days to measure viral-yield inhibition. Thecells (72 d.p.i.) were fixed with 80% acetone and were analyzed by anIFA assay using an antibody against HCoV-OC43 N protein. EC₅₀ weredetermined accordingly. The viabilities of HCT-8 cells were also studiedusing CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit(“MTS”) (Promega, Madison, WI, USA). CC₅₀ values were calculated. Inaddition, to demonstrate the cytopathic effect of HCT-8 infected byHCoV-OC43 at an MOI of 0.05, the cells thus treated were stained withcrystal violet after fixation at 6 d.p.i.

Compounds 1-6 exhibited strong antiviral activity against HCoV-OC43viruses, having EC₅₀ values ranging from 16 nM to 1.8 μM for HCoV-OC43with a selectivity index from 610 to 5.3 (Table 2).

TABLE 2 Compound EC₅₀ (nM) CC₅₀ (nM) Selectivity index 1 68.0 ±2.7 >10000 >147 2 16.4 ± 4.7 >10000 >610 3 55.9 ± 6.2 >10000 >179 4 51.7± 6.1 >10000 >193 5 213 ± 62 >10000 >46.9 6 1892 ± 228 >10000 >5.3^(a)Data means ± S.D. from three independent experiments, each induplicate (HCoV-OC43). ^(b)EC₅₀: The values of 50% maximal effectiveconcentration. ^(c)CC₅₀: The values of 50% maximal cytotoxicconcentration.

Notably, Compounds 1-3 each had a very high potency inhibiting HCoV-OC43viruses. These three compounds were then evaluated for cytopathiceffects at 6 d.p.i. and IFA at 3 d.p.i., followed by a subsequent endpoint assay and TCID50 determination to show inhibitory activities in adose-dependent manner. In the end point assay, HTC-8 cells were infectedwith HCoV-OC43 viruses at a MOI of 0.05 for 72 hours to reach a viralyield of 10⁷ p.f.u./ml and then treated using one of Compounds 1-3 at apredetermined concentration. Compounds 1-3 each significantly blockedviral replication and decreased viral yields, resulting in a viral yieldreduction by 7 orders of magnitude at a concentration between 30 nM and300 nM. See FIG. 1 .

Example 3: Inhibition of HCoV-229E Viruses

Compounds 1-3 were used to inhibiting HCoV-229E virus, another humancoronavirus belonging to genus of the alpha-coronavirus.

HCoV-229E viral genome replication was examined by RT-PCR with specificprimers against its open reading frame 1 (“ORF1”) and ORF nucleocapsid(“ORFN”). See Yang et al., Front. Pharmacol. 11, Article 606097 (2020).

Fetus lung fibroblast MRC5 cells were inoculated with the HCoV-229Evirus at a MOI of 1. The MRC5 cells thus infected were treated with oneof Compounds 1-3. Untreated MRC5 cells were used as a control sample.RT-PCR analysis was performed. Compounds 1-3 each inhibited HCoV-229Eviral genome replication and subgenomic viral RNA syntheses at aconcentration between 10 nM and 300 nM.

Cytopathic effects were studied and visualized after the infected cellswere treated with one of Compounds 1-3. An end point assay by TCID50 wasperformed. Crystal violet was used to stain live cells to determine theEC₅₀ of each compound. See Table 3. Further, CC₅₀ and selective indexwere calculated.

TABLE 3 Compound EC₅₀ (nM) CC₅₀ (nM) Selectivity index 1 71.4 ± 13.26635 ± 578 93 2 6.5 ± 1.0 1712 ± 161 264 3 25.4 ± 3.1  7362 ± 288 290Data means ± S.D. from three independent experiments, each in duplicate(HCoV-229E). EC₅₀: The values of 50% maximal effective concentration.CC₅₀: The values of 50% maximal cytotoxic concentration.

HCoV-229E viral yield was studied following the procedure describedabove. Compounds 1-3 each significantly blocked viral replication andabolished the viral yield, resulting in a reduction of viral yield by6-7 orders of magnitude at concentrations in the range of 30 nM to 600nM in HCoV-229E infected MRC5 cells. EXAMPLE 4: Inhibition of Zika virus

Compounds 1-9 were evaluated for their inhibitory activities againstZika virus using an IFA assay, which targeted Zika virus Envelope (“E”)protein.

Vero E6 cells (BCRC #60476, Bioresource Collection and Research Center,Hsinchu, Taiwan) were treated with one of Compounds 1-9 at apredetermined concentration for 1 hour at 37° C. The cells were adsorbedwith Zika virus (ATCC VR-1843, American Type Culture Collection,Manassas, VA, USA) at MOI=0.02 for 49 hours at 37° C. The cells werefixed with 10% formalin, permeabilized with methanol. The cells werestained with anti-flavivirus E antibody (ATCC HB-112, American TypeCulture Collection, Manassas, VA, USA) and anti-mouse IgG-FITC (MPBiomedicals, Irvine, CA, USA). The nuclei were counter stained with DAPI(in blue) (Thermo Fisher Scientific, MA, USA). The E protein expressionwas measured using a high-content image analysis system (MolecularDevices). The cell viability was determined by MTS. Both EC₅₀ and CC₅₀were calculated by Prism software.

Their cytotoxic effects were also determined by the MTS assay describedabove using Vero E6 cells. Each of Compounds 1-9 effectively inhibitedZika virus. On the other hand, their 50% cytotoxic concentrations weremuch higher than their EC₅₀, indicating a high selectivity of inhibitingZika virus. The results were shown in Table 4 below.

TABLE 4 Compound EC₅₀ (nM) CC₅₀ (nM) 1 61.4 >5000 2 15.3 >5000 315.9 >5000 4 13.3 >5000 5 156 >5000 6 1036 >5000 7 44.4 >5000 8304 >5000 9 144 >5000

Example 5: Inhibition of Dengue Virus

Compounds 1-9 were evaluated for their inhibitory activities againstDengue virus, another single positive-stranded RNA virus belonging togenus of flavivirus, using an IFA assay that targeted the Dengue virusEnvelope (“E”) protein.

Vero E6 cells (BCRC #60476, Bioresource Collection and Research Center,Hsinchu, Taiwan) were treated with one of Compounds 1-9 at apredetermined concentration for 1 hour at 37° C. The cells were infectedwith Dengue virus type 1 (“DV1”, ATCC VR-1856, American Type CultureCollection, Manassas, VA, USA) at MOI=0.002 or Dengue virus type 2(“DV2”, ATCC VR-1584, American Type Culture Collection, Manassas, VA,USA) at MOI=0.02, both for 5 days at 33° C. They were then fixed with10% formalin and permeabilized with methanol. Subsequently, the cellswere stained with anti-flavivirus E antibody (ATCC HB-112, American TypeCulture Collection, Manassas, VA, USA) and anti-mouse IgG-FITC (MPBiomedicals, Irvine, CA, USA). The nuclei of the cells were counterstained with DAPI (Thermo Fisher Scientific, MA, USA). The E proteinexpression was measured by using a high-content image analysis system(Molecular Devices). The cell viability was determined by MTS. EC50 andCC50 were calculated by Prism software.

Compounds 1-9 each were highly effective in inhibiting dengue virus. SeeTable 5 below.

TABLE 5 DV1 DV2 Vero E6 compound EC₅₀ (nM) EC₅₀ (nM) CC₅₀ (nM) 1 18.432.5 >5000 2 4.3 4.7 >5000 3 19.6 29 >5000 4 7.6 8.3 >5000 5 131286 >5000 6 1638 2641 >5000 7 73.1 124 >5000 8 247 617 >5000 9 296 616>5000

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

Further, from the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A method of treating a disease caused by apositive strand RNA virus, the method comprising: identifying a subjectsuffering from the disease, and administering to the subject aneffective amount of a compound of Formula I:

wherein each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, and R₁₅, independently, is H, halo, alkyl, aryl, cycloalkyl,heteroaryl, heterocycloalkyl, OH, alkoxy, carbonyloxy, or amino; each ofR₁₆ and R₁₇, independently, is H, halo, alkyl, aryl, cycloalkyl,heteroaryl, heterocycloalkyl, OH, alkoxy, carbonyloxy, or amino, or R₁₆and R₁₇ together are a single bond; each of

and

, independently, is a single bond or a double bond; and n is 1, 2 or 3,provided that when

is a single bond, X is C═O or CR′R″ and Y is N or N⁺→O⁻, and when

is a double bond, X is CR′, Y is N⁺, and a counterion coexists in thecompound, each of R′ and R″, independently, being H, halo, alkyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, OH, alkoxy, or amino.
 2. Themethod of claim 1, wherein the positive strand RNA virus is a flavivirusor a coronavirus.
 3. The method of claim 1, wherein the positive strandRNA virus is a SARS-CoV-2 virus, a Zika virus, a Dengue virus, aHCoV-OC43 virus, or a HCoV-229E virus.
 4. The method of claim 2, whereinthe positive strand RNA virus is a Zika virus or a Dengue virus.
 5. Themethod of claim 2, wherein the positive strand RNA virus is a SARS-CoV-2virus.
 6. The method of claim 1, wherein each of R₁, R₅, R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅ is H.
 7. The method of claim 1, wherein eachof R₂, R₃, R₆, and R₇, independently, is alkoxy.
 8. The method of claim1, wherein R₄ is H or alkoxy.
 9. The method of claim 1, wherein R₁₆ andR₁₇ together are a single bond.
 10. The method of claim 1, wherein X isCH₂.
 11. The method of claim 1, wherein Y is N or N+→O—.
 12. The methodof claim 1, wherein each of

and

is a single bond.
 13. The method of claim 1, wherein n is 1 or
 2. 14.The method of claim 6, wherein each of R₂, R₃, R₆, and R₇,independently, is methoxy and R₄ is H or alkoxy.
 15. The method of claim14, wherein R₁₆ and R₁₇ together are a single bond, X is CH₂, and Y is Nor N+→O—.
 16. The method of claim 1, wherein each of R₁, R₅, R₈, R₉,R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅ is H; each of R₂, R₃, R₆, and R₇,independently, is alkoxy; R₄ is H or alkoxy; R₁₆ and R₁₇ together are asingle bond; X is CH₂; Y is N or N+→O—; each of

and

is a single bond; and n is 1 or
 2. 17. The method of claim 1, whereinthe compound of Formula I is selected from the group consisting ofCompound 1, Compound 4, and Compound 6:


18. The method of claim 17, wherein the positive strand RNA virus is aSARS-CoV-2 virus, a Zika virus, a Dengue virus, a HCoV-OC43 virus, or aHCoV-229E virus.
 19. The method of claim 17, wherein the positive strandRNA virus is a Zika virus or a Dengue virus.
 20. The method of claim 17,wherein the positive strand RNA virus is a SARS-CoV-2 virus.